Abstract INTRODUCTION Glioblastoma (GBM) is the most common malignant brain tumor in adults and is associated with a dismal prognosis. Neoadjuvant anti-PD-1 blockade has demonstrated efficacy in melanoma, non-small cell lung cancer and recurrent GBM; however, responses vary. While T cells have garnered considerable attention in the context of immunotherapy, the role of myeloid cells in the GBM microenvironment remains controversial. METHODS We isolated CD45+ immune populations from patients who underwent brain tumor resection at UCLA. We hypothesized that myeloid cells in glioblastoma contribute to T cell dysfunction; however, this immune suppression can be mitigated by neoadjuvant PD-1 inhibition. To test this, we utilized mass cytometry and single-cell RNA sequencing to characterize these immune populations. RESULTS Mass cytometry profiling of tumor infiltrating lymphocytes from patients with GBM demonstrated a preponderance of CD11b+ myeloid populations (75% versus 25% CD3+). At the transcriptomic level, myeloid cells in newly diagnosed GBMs exhibited decreased expression of CCL4 (loge fold change -1.18, Bonferroni-adjusted P = 1.62x10-254) and its ligands compared to anaplastic astrocytoma. In ranked gene set enrichment analysis, patients who received neoadjuvant pembrolizumab demonstrated enrichment in TNFα-, NFκB- and lipid metabolism-related gene sets by bootstrapped Kolmogorov-Smirnov test (Benjamini-Hochberg adjusted P = 4.74x10-3, 1.45x10-2 and 2.48x10-3, respectively) in tumor-associated myeloid populations. Additionally, single-cell trajectory analysis demonstrated increased CCL4 and decreased ISG15 with neoadjuvant checkpoint inhibition. CONCLUSIONS Here, we utilize mass cytometry and single-cell RNA sequencing to demonstrate the predominance and transcriptomic features of myeloid populations in GBM. Myeloid cells in patients who receive neoadjuvant PD-1 blockade re-express increased levels NFκB, TNFα and CCL4, a cytokine crucial for the recruitment of dendritic cells to the tumor for antigen-specific T cell activation. By delving into the GBM microenvironment at the single-cell level, we hope to better delineate the role of myeloid populations in this uniformly fatal tumor.